Methods for producing genetically modified plants, plant materials and plant products produced thereby

ABSTRACT

Methods for producing genetically modified plants, particularly woody plants, and most particularly plants of the Eucalyptus and Pinus species, involve transformation of target plant material with a desired genetic construct and regeneration of the transformed plant material using an adventitious shoot bud system. The methods provide a high transformation efficiency and substantially reduce the duration of the transformation and regeneration protocols. Stem segments of a target plant are transformed using Agrobacterium-mediated techniques, and adventitious shoot buds are regenerated from the Agrobacterium-infected stem segments. Preferred culture media, including selection media, and improved plant culture techniques are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to International PatentApplication No. PCT/NZ99/00155, filed Sep. 15, 1999, and is acontinuation-in-part of U.S. application Ser. No. 09/153,320, filed Sep.15, 1998.

TECHNICAL FIELD OF THE INVENTION

[0002] This invention relates to methods for producing geneticallymodified plants, particularly woody plants, and most particularly plantsof the Eucalyptus and Pinus species, as well as to plants, plantmaterials and plant products produced by or from such geneticmodification. This invention relates, more specifically, to techniquesfor producing genetically modified plants, including transgenic woodyplants and interspecies hybrid woody plants, particularly of theEucalyptus and Pinus species.

BACKGROUND OF THE INVENTION

[0003] Recent advances in plant genetic engineering have made possiblethe transfer of DNA into plants, including commercially importantforestry tree species. The application of genetic engineering tocommercially important forestry varieties provides opportunities toincorporate new or improved traits of commercial interest, such asdisease resistance, male sterility, increased productivity, rootingability, wood quality, and others, in forestry varieties.

[0004] Commercial scale planting stocks of forestry varieties aregenerally produced directly from seed or from rooted cuttings. In bothof these production systems, traditional plant-breeding techniques areused to produce superior planting stock. The application of geneticengineering techniques to stably incorporate homologous and/orheterologous genetic material into plants offers the potential ofimproved planting stocks compared to those developed using traditionalbreeding techniques.

[0005] The overall efficiency of techniques for genetically modifyingplants depends upon the efficiency of the transformation technique(s)used to stably incorporate the homologous and/or heterologous geneticmaterial into plant cells or tissues, and the regeneration technique(s)used to produce viable plants from transformed cells. In general, theefficiency of transformation and regeneration techniques adapted forgenetically modifying forestry plants, such as plants of the Eucalyptusspecies, is low.

[0006] Publications report the successful transfer of DNA intocommercial varieties of tree species, including Eucalyptus. “Prospectsfor Eucalyptus transformation,” Biological Sciences Symposium, TAPPIPress: pp.313-326, 1997. Genetic transformation has generally beenachieved through Agrobacterium-mediated transformation. Transformationtechniques have been demonstrated using reporter genes such as GUS(β-D-glucuronidase), nptII (neomycin phosphotransferase) and cat(chloramphenicol acetyl transferase). A reproducible and reliable tissueculture regeneration system is required for regenerating plants fromtransformed cells. Regeneration systems developed for use with forestryvarieties have generally demonstrated very low levels of reproducibilityand efficiency. Regeneration is generally the limiting factor in theproduction of transgenic forestry species.

[0007] Techniques for plant tissue culture have been developed and usedextensively for micropropagation of various Eucalyptus species. LeRouxand Van Staden, “Micropropagation and tissue culture of Eucalyptus: areview,” Tree Physiology 9:435-477, 1991. Techniques used formicropropagation generally involve axillary bud multiplication. Theaxillary bud is induced from the leaf axils of stem segments, the bud isallowed to elongate into a shoot, and it is then allowed to multiply inthe same manner, producing more axillary shoots. When sufficient copiesof a clone are produced, the shoots are rooted and then transplanted.This system has been widely used for commercial production of clones forreforestation because it reliably produces stably cloned propagules thatare true to type.

[0008] Although the axillary bud multiplication system is welldeveloped, it is not a preferred regeneration system for regeneratinggenetically modified plants. Transgenic plants produced using axillarybud multiplication regeneration techniques are often chimeric becausethe axillary buds are generated from preformed buds that may carry amixture of transformed and non-transformed cells. Only portions oftransgenic plants produced from chimeric tissues are transformed andcarry the introduced genetic material.

[0009] Applicants are aware of two published protocols for regenerationof Eucalyptus. In a protocol published in Plant Cell Reports 13:473-476,1994, an organogenesis pathway using leaf explants was described. In aprotocol published in Suid-Afrikaanse Bosboutydskrif 157:59-65, 1991, asomatic embryogenesis pathway using leaf explants was described. Thesereported systems demonstrated a low efficiency and, additionally,required an impractically long time period for the regeneration process.The long duration (six months) of the regeneration process is notcommercially feasible. Furthermore, neither of these systems wassuccessfully reproduced by applicants.

[0010] The success and efficiency of methods for producing geneticallymodified plants thus depends on the selection and optimization of atissue culture regeneration system that provides de novo origination ofplant material from transformed cells, and development of thegenetically modified plant material to produce a genetically modifiedplant. Techniques developed to date for genetically modifying forestryspecies such as Eucalyptus generally demonstrate low reproducibility ofthe regeneration protocol, long duration of regeneration, low efficiencyof plant regeneration (0-5%), and low transformation efficiency. Thepresent invention is directed to improved methods for producinggenetically modified plants, particularly forestry species, and mostparticularly plants of the Eucalyptus and Pinus species.

SUMMARY OF THE INVENTION

[0011] The present invention involves methods for producing geneticallymodified plant material, particularly woody plant material of theEucalyptus or Pinus species. Agrobacterium-mediated transformationtechniques, whereby one or more genetic construct(s) comprising areporter gene and the genetic material desired to be introduced istransformed into an Agrobacterium strain using well-known techniques,are preferred. The target plant material is inoculated withAgrobacterium carrying the genetic construct of interest.

[0012] Preferred tissue explants of the target plant comprise stemsegments from micropropagated shoot cultures. The target plant stemsegments may be pretreated in a multiplication medium and thentransferred to a shoot elongation medium to promote formation of matureshoots. Nodes may be excised from the target plant stem segments andleaves from the stem segments and/or selected nodes of the shootexplants are preferably removed. The stem segments and/or nodes may beadditionally wounded, such as by puncturing or cutting. The stemsegments and/or nodes may then be incubated with a transformedAgrobacterium culture to inoculate the target plant explants with thedesired genetic material. Following inoculation, regeneration ofadventitious shoot buds from the Agrobacterium infected stem segments ispromoted in tissue culture using a combination of regeneration agents.The system of the present invention is advantageous for producinggenetically modified plants because it employs transformation andregeneration techniques that provide de novo shoot origination fromtransformed cells.

[0013] Following a suitable period for adventitious shoot bud formation,putative transformed adventitious shoots may be excised from the stemsegments. Selection techniques may then be used to identify successfullytransformed adventitious shoot buds. The selection technique may vary,depending upon the reporter construct used. According to preferredembodiments, the reporter construct introduced to the Agrobacterium and,then, the explants, includes an antibiotic-resistance gene. In thissystem, suitable selection agents comprise antibiotic agents. A twostage selection technique is preferably employed, whereby theadventitious shoot buds are exposed to a first selection medium having afirst concentration of the selection agent, preferably an antibiotic,and the surviving adventitious buds are then exposed to a secondselection medium having a second concentration of the selection agent,the second selection agent concentration being greater than the firstselection agent concentration. This two stage selection techniquesubstantially eliminates the presence of chimeric shoots in the selectedadventitious shoot buds.

[0014] Following selection of transformed adventitious shoots, thetransformed adventitious shoot buds are transferred to a rooting mediumand roots are generated using techniques that are well known in the art.Rooted shoots, or plantlets, may then be transferred to planting mediumand planted to complete the transformation and regeneration procedure.The plantlets include the genetic material introduced using the geneticconstruct. Genetically modified plantlets may be grown to geneticallymodified mature plants. The products obtained from genetically modifiedmature plants, such as timber, wood pulp, fuel wood, and the like, alsocontain the genetic modification.

[0015] The transformation and regeneration methods of the presentinvention are reproducible and substantially reduce the duration oftransformation and regeneration of genetically modified plant materialscompared to methods previously reported for forestry plant species.Applied to the Eucalyptus species, methods of the present inventionreduce the time required for transformation and regeneration from sixmonths or more to about ten to fifteen weeks. This reduction issubstantial. The methods of the present invention are suitable forcommercial production of genetically modified plants, including forestryspecies such as Eucalyptus and Pinus.

[0016] The methods of the present invention for producing geneticallymodified plants and plant materials are especially suitable for use withforestry species, particularly Eucalyptus and Pinus species. Thesemethods may provide the introduction of new genes, additional copies ofexistent genes, or non-coding portions of a genome, into selected cloneswith little disturbance of the plant's genome. Genetic material thatproduces desirable traits, such as insect tolerance, disease resistance,herbicide tolerance, male sterility, rooting ability, cold tolerance,drought tolerance, salinity tolerance, and modification of woodproperties and growth rates and properties, and the like, may beintroduced. The genetic material introduced may be homologous orheterologous to the genome of the target plant.

[0017] The present invention also contemplates plants, plant materials,and plant products derived from genetically modified plants producedaccording to methods of the present invention. Plants include mature andimmature plants grown from plantlets produced according to methods ofthe present invention, as well as progeny of such plants and plantspropagated using materials from such plants. Plant materials includeplant cells or tissues such as seeds, flowers, bark, stems, etc. of allsuch plants. Plant products include any materials derived from plantmaterials, such as wood products, pulp products, and the like.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0018] Using the methods and materials of the present invention, thegenome of a target plant may be modified by incorporating homologous orheterologous genetic material. Additional copies of genes encodingcertain polypeptides, or functional portions of certain polypeptides,such as enzymes involved in a biosynthetic pathway, may be introducedinto a target plant using the methods of the present invention toincrease the level of a polypeptide of interest. Similarly, a change inthe level of a polypeptide of interest in a target plant may be achievedby transforming the target plant with antisense copies of genes encodingthe polypeptide of interest, or a functional portion of the polypeptideof interest. Additionally, the number of copies of genes encodingdifferent polypeptides, such as enzymes in a biosynthetic pathway, maybe manipulated to modify the relative amount of each polypeptidesynthesized, leading to the formation of an end product having amodified composition. Non-coding portions of polynucleotides, such asregulatory polynucleotides and polynucleotides encoding regulatoryfactors, such as transcription factors, and/or functional portions oftranscription factors, and/or antisense copies of such regulatoryfactors, may also be introduced to target plant material to modulate theexpression of certain polypeptides. These materials are exemplary of thetypes of genetic material suitable for modifying the genome of targetplant material. Numerous other materials may also be introduced.

[0019] The methods of the present invention preferably employ shootcultures of the target plant material as a starting material.Micropropagated shoot cultures may be generated by surface sterilizingyoung shoots from field grown juvenile and mature stage target plants ina sterilization medium, rinsing the sterilized shoots, and then exposingthem to a multiplication or elongation medium. Suitable sterilizationmedia, such as 0.01% mercuric chloride solution, are known, and repeatedrinsing may be performed with sterile, distilled water. Alternatively,micropropagated shoot cultures may be obtained from forestry companies.

[0020] According to preferred embodiments, in vitro micropropagatedshoot cultures are grown for a period of from one week to several weeks,preferably three weeks, on a multiplication medium. A preferredmultiplication medium comprises full strength MS (Murashige and Skoog)medium (Sigma M5519), sucrose, Benzylaminopurine (BA), and NaphthaleneAcetic Acid (NAA). The multiplication medium preferably comprisessucrose at a concentration of 30 g/l, BA at a concentration of 0.1 mg/l,and NAA at a concentration of 0.01 mg/l in full strength MS medium.

[0021] The shoot cultures may then preferably be transferred to a shootelongation medium. The shoot elongation medium additionally comprises aplant growth promoter, such as gibberellic acid, at a concentration ofabout 1 mg/l. Shoot cultures are preferably exposed to the shootelongation medium for at least three weeks, more preferably for four tosix weeks. Shoot cultures are preferably subcultured to fresh mediumevery two to four weeks, and are preferably transferred to fresh mediumabout two to three weeks before transformation. Shoots of the targetplant material are preferably allowed to grow to a size of from 1 to 8cm in length, more preferably from about 3 to 4 cm in length, beforethey are transformed to incorporate the desired genetic material.

[0022] Unless otherwise noted, in vitro cell culture conditionspreferably include a 16 hour photoperiod using cool white fluorescentlighting and temperatures of about 20° C. Cultures are preferably grownin Petri dishes, with multiple shoots per Petri dish, and with theshoots arranged horizontally.

[0023] The “genetic material” transformed into the target plant materialincludes one or more genetic construct(s) comprising one or morepolynucleotide(s) desired to be introduced to the target plant material,and a reporter construct. Genetic constructs introduced into the targetplant material may comprise genetic material that is homologous and/orheterologous to the target plant material, and may includepolynucleotides encoding a polypeptide or a functional portion of apolypeptide, polynucleotides encoding a regulatory factor, such as atranscription factor, non-coding polynucleotides such as regulatorypolynucleotides, and antisense polynucleotides that inhibit expressionof a specified polypeptide. The genetic construct may additionallycomprise one or more regulatory elements, such as one or more promoters.The genetic construct is preferably functional in the target plant.

[0024] According to one embodiment, the genetic constructs used inconnection with the present invention include an open reading framecoding for at least a functional portion of a polypeptide of interest inthe target plant material. A polypeptide of interest may be a structuralor functional polypeptide, or a regulatory polypeptide such as atranscription factor. As used herein, the “functional portion” of apolypeptide is that portion which contains the active site essential foraffecting the metabolic step, i.e. the portion of the molecule that iscapable of binding one or more reactants or is capable of improving orregulating the rate of reaction. The active site may be made up ofseparate portions present on one or more polypeptide chains and willgenerally exhibit high substrate specificity.

[0025] A target plant may be transformed with more than one geneticconstruct of the present invention, thereby modulating a biosyntheticpathway for the activity of more than one polypeptide, affecting anactivity in more than one tissue or affecting an activity at more thanone expression time. Similarly, a genetic construct may be assembledcontaining more than one open reading frame coding for a polypeptide ormore than one non-coding region of a gene.

[0026] The word “polynucleotide(s),” as used herein, means a polymericcollection of nucleotides and includes DNA and corresponding RNAmolecules, both sense and anti-sense strands, and comprehends cDNA,genomic DNA and recombinant DNA, as well as wholly or partiallysynthesized polynucleotides. A polynucleotide may be an entire gene, orany portion thereof. Operable anti-sense polynucleotides may comprise afragment of the corresponding polynucleotide, and the definition of“polynucleotide” includes all such operable anti-sense fragments.

[0027] Identification of genomic DNA and heterologous species DNA can beaccomplished by standard DNA/DNA hybridization techniques, underappropriately stringent conditions, using all or part of a cDNA sequenceas a probe to screen an appropriate library. Alternatively, PCRtechniques using oligonucleotide primers that are designed based onknown genomic DNA, cDNA and protein sequences can be used to amplify andidentify genomic and cDNA sequences. Synthetic DNA corresponding to theidentified sequences and variants may be produced by conventionalsynthetic methods. All of the polynucleotides described herein areisolated and purified, as those terms are commonly used in the art. Apolynucleotide of interest, as used herein, is a polynucleotide that ishomologous or heterologous to the genome of the target plant and altersthe genome of the target plant.

[0028] As used herein, the term “polypeptide” encompasses amino acidchains of any length, including full length proteins, wherein amino acidresidues are linked by covalent peptide bonds.

[0029] When the genetic construct comprises a coding portion of apolynucleotide, the genetic construct further comprises a gene promotersequence and a gene termination sequence operably linked to thepolynucleotide to be transcribed. The gene promoter sequence isgenerally positioned at the 5′ end of the polynucleotide to betranscribed, and is employed to initiate transcription of thepolynucleotide. Promoter sequences are generally found in the 5′non-coding region of a gene but they may exist in introns or in thecoding region. When the construct includes an open reading frame in asense orientation, the gene promoter sequence also initiates translationof the open reading frame. For genetic constructs comprising either anopen reading frame in an antisense orientation or a non-coding region,the gene promoter sequence may comprise a transcription initiation sitehaving an RNA polymerase binding site.

[0030] A variety of gene promoter sequences which may be usefullyemployed in the genetic constricts of the present invention are wellknown in the art. The promoter gene sequence, and also the genetermination sequence, may be endogenous to the target plant host or maybe exogenous, provided the promoter is functional in the target host.For example, the promoter and termination sequences may be from otherplant species, plant viruses, bacterial plasmids and the like.

[0031] Factors influencing the choice of promoter include the desiredtissue specificity of the construct, and the timing of transcription andtranslation. For example, constitutive promoters, such as the 35SCauliflower Mosaic Virus (CaMV 35S) promoter, will affect the activityof a polypeptide in all parts of the plant. Use of a tissue specificpromoter will result in production of the desired sense or antisense RNAonly in the tissue of interest. With genetic constructs employinginducible gene promoter sequences, the rate of RNA polymerase bindingand initiation may be modulated by external stimuli, such as light,heat, anaerobic stress, alteration in nutrient conditions and the like.Temporally regulated promoters may be employed to effect modulation ofthe rate of RNA polymerase binding and initiation at a specific timeduring development of a transformed cell. Preferably, the originalpromoters from the enzyme gene in question, or promoters from a specifictissue-targeted gene in the organism to be transformed, such asEucalyptus or Pinus, are used. Other examples of gene promoters whichmay be usefully employed in the present invention include mannopinesynthase (mas), octopine synthase (ocs) and those reviewed by Chua etal. (Science, 244:174-181, 1989). Multiple copies of promoters, ormultiple promoters, may be used to selectively stimulate expression of apolynucleotide comprising a part of the genetic construct.

[0032] The gene termination sequence, which is located 3′ to the DNAsequence to be transcribed, may come from the same gene as the genepromoter sequence or may be from a different gene. Many gene terminationsequences known in the art may be usefully employed in the presentinvention, such as the 3′ end of the Agrobacterium tumefaciens nopalinesynthase gene. However, preferred gene terminator sequences are thosefrom the original polypeptide gene, or from the target species beingtransformed.

[0033] The genetic constructs of the present invention also comprise areporter gene or a selection marker that is effective in target plantcells to permit the detection of transformed cells containing thegenetic construct. Such reporter genes and selection markers, which arewell known in the art, typically confer resistance to one or moretoxins. A chimeric gene that expresses β-D-glucuronidase (GUS) intransformed plant tissues but not in bacterial cells is a preferredselection marker for use in methods of the present invention. The binaryvector pKIWI 105, constructed as described by Janssen and Gardner inPlant Molecular Biology 14:61-72, 1989, is an especially preferredselection marker. The preferred selection marker may be modified toprovide multiple copies of a desired promoter, such as the Cauliflowermosaic virus 355 promoter. Plant material expressing GUS is resistant toantibiotics such as kanamycin. Another suitable marker is the nptIIgene, whose expression results in resistance to kanamycin or hygromycin,antibiotics which are generally toxic to plant cells at a moderateconcentration (Rogers et al. in Weissbach A and Weissbach H, eds.,Methods for Plant Molecular Biology, Academic Press Inc.: San Diego,Calif., 1988). Alternatively, the presence of the desired construct intransformed cells may be determined by means of other techniques thatare well known in the art, such as Southern and Western blots.

[0034] Techniques for operatively linking the components of the geneticconstructs used to transform target plant materials are well known inthe art and include the use of synthetic linkers containing one or morerestriction endonuclease sites as described, for example, by Sambrook etal. (Molecular cloning: a laboratory manual, CSHL Press: Cold SpringHarbor, N.Y., 1989). Genetic constructs used in methods of the presentinvention may be linked to a vector having at least one replicationsystem, for example, E. coli, whereby after each manipulation, theresulting construct can be cloned and sequenced and the correctness ofthe manipulation determined.

[0035] For applications where amplification of a polypeptide is desired,an open reading frame encoding the polypeptide of interest, or apolynucleotide encoding a regulatory factor that modulates expression ofthe polypeptide of interest, may be inserted in the genetic construct ina sense orientation, such that transformation of a target plant with thegenetic construct will produce an increase in the number of copies ofthe gene or an increase in the expression of the gene and, consequently,an increase in the amount of the polypeptide. When down-regulation of apolypeptide is desired, an open reading frame encoding the polypeptideof interest may be inserted in the genetic construct in an antisenseorientation, such that the RNA produced by transcription of thepolynucleotide is complementary to the endogenous mRNA sequence. This,in turn, will result in a decrease in the number of copies of the geneand therefore a decrease in the amount of enzyme. Alternatively,modulation may be achieved by inserting a polynucleotide encoding aregulatory element, such as a promoter or a transcription factor, thatmodulates expression of the polynucleotide encoding the polypeptide ofinterest.

[0036] In another embodiment, the genetic construct used to transformthe target plant material may comprise a nucleotide sequence including anon-coding region of a gene coding for a polynucleotide of interest, ora nucleotide sequence complementary to such a non-coding region. As usedherein the term “non-coding region” includes both transcribed sequenceswhich are not translated, and non-transcribed sequences within about2000 base pairs 5′ or 3′ of the translated sequences or open readingframes. Examples of non-coding regions which may be usefully employed inthe inventive constructs include introns and 5′-non-coding leadersequences. Transformation of a target plant with such a geneticconstruct may lead to a reduction in the amount of a selectedpolypeptide synthesized by the plant by the process of cosuppression, ina manner similar to that discussed, for example, by Napoli et al. (PlantCell 2:279-290, 1990) and de Carvalho Niebel et al. (Plant Cell7:347-358, 1995).

[0037] Genetic constructs may be used to transform a variety of plantsusing the methods of the present invention, including monocotyledonous(e.g., grasses, corn, grains, oat, wheat and barley), dicotyledonous(e.g., Arabidopsis, tobacco, legumes, alfalfa, oaks, Eucalyptus, maple),and Gymnosperms (e.g., Scots pine (Aronen, Finnish Forest Res. Papers,Vol. 595, 1996), white spruce (Ellis et al., Biotechnology 11:84-89,1993), and larch (Huang et al., In vitro Cell 27:201-207, 1991). Inpreferred embodiments, the genetic constructs are employed to transform“woody plants,” which are herein defined as a tree or shrub whose stemlives for a number of years and increases in diameter each year by theaddition of woody tissue. The target plant is preferably selected fromthe group consisting of the Eucalyptus and Pinus species, mostpreferably from the group consisting of Eucalyptus grandis and Pinusradiata.

[0038] Techniques for stably incorporating genetic constructs into thegenome of target plants are well known in the art and includeAgrobacterium-mediated introduction, electroporation, protoplast fusion,injection into reproductive organs, injection into immature embryos,high velocity projectile introduction, and the like. The choice oftechnique will depend upon the target plant to be transformed. Forexample, dicotyledonous plants and certain monocots and gymnosperms maybe transformed by Agrobacterium Ti plasmid technology as described, forexample by Bevan (Nucleic Acids Res. 12:8711-8721, 1984). Targets forthe introduction of the genetic constructs of the present inventioninclude tissues, such as leaf tissue, disseminated cells, protoplasts,seeds, embryos, meristematic regions, cotyledons, hypocotyls, and thelike. Preferred target plant materials for transformation according tomethods of the present invention include in vitro micropropagated shootcultures prepared as described above.

[0039] Transfer of one or more genetic constructs into target plantshoots is preferably accomplished using Agrobacterium-mediatedtransformation techniques. Numerous Agrobacterium strains are suitableand are commercially available. Agrobacterium tumefaciens strain AGL1(Biotechnology 9:963-967, 1991) is available and is a preferredAgrobacterium strain. Methods for transforming a population of theAgrobacterium strain with a genetic construct are well known. The freezethaw method described in An et al., “Binary Vectors,” in Gelvin SB andSchilperoort RA, eds, Plant Molecular Biology Manual, Dordrecht: KluwerAcademic Publishers, pp. A3/1-A3/19, 1988, is a preferred method fortransforming the Agrobacterium culture with the genetic construct ofinterest.

[0040] According to preferred embodiments, colonies of Agrobacteriumcarrying the genetic construct of interest are prepared for inoculationof the target plant material according to the following techniques.Agrobacterium colonies are grown on a growth medium such as YEP mediumcomprising yeast, peptone and sodium chloride. According to especiallypreferred embodiments, the growth medium comprises yeast at aconcentration of 20 g/l, peptone at a concentration of 20 g/l and sodiumchloride at a concentration of 10 g/l. A single colony from the platemay be selected and grown in a culture medium comprising a selectionagent for the selection marker. Suitable selection agents comprise, forexample, antibiotics. According to an especially preferred embodimentusing an Agrobacterium culture transformed with a genetic constructcomprising a chimeric GUS gene, the selection agent is kanamycin. Theselected Agrobacterium colony is preferably grown in YEP mediumcomprising kanamycin and rifampicin. Preferred medium comprises 100 mg/lkanamycin and 50 mg/l rifampicin. Cultures may be incubated at 29° C.with vigorous shaking for several hours. The culture may then becentrifuged, washed, and resuspended in medium such as an MS mediumcomprising acetosyringone at a concentration of 50 μM. The inoculum ispreferably adjusted to an OD₆₀₀ of about 0.15, and cultured on a shakerfor several hours at 29° C. before inoculation.

[0041] Mature shoots of the target plant material prepared as describedabove are selected for transformation. Stem segments from each node areexcised. Stem segments from the second and third nodes are preferred foruse in methods of the present invention. All leaves are preferablypeeled from the stems, and additional wounding may be inflicted toenhance the efficacy and efficiency of Agrobacterium inoculation.Wounding of the stems segments preferably takes place in proximity tothe axillary region of each node, such as in areas adjacent to andsurrounding the axillary region of each node. Wounding involves exposingcells below the exterior cellular layer of stem segments to provideaccess to externally supplied gases or liquids. Wounding may involvepuncturing the exterior cellular layer of stem segments using a needleor another sharp implement, or may be achieved, for example, by lightlongitudinal cutting or scoring of the stem, such as with a scalpelblade. According to a preferred embodiment, a pre-induction procedure isused whereby the target plant nodes are placed in bud induction mediumprior to wounding, and prior to Agrobacteriaum inoculation, to permitsome axillary shoot growth prior to wounding and inoculation. Theselected stem segments, preferably including the second and third nodes,are inoculated with the Agrobacterium culture prepared as describedabove.

[0042] Inoculation of stem segments with the Agrobacterium suspensiontakes place under conditions that optimize infection of the stemsegments. Incubation may be continued for at least about twenty minutes,more preferably about thirty minutes, on a shaker at a temperature ofabout 20° C. According to a preferred embodiment, reduced pressureconditions are applied during at least a portion of the inoculationperiod. That is, during inoculation, the pressure in the areasurrounding the stem segments and Agrobacterium suspension is reduced toa pressure less than that of the standard, ambient atmosphere.Application of a vacuum, for example, for at least a portion of theinoculation period, may improve transformation efficiency. Applicationof a vacuum for at least about three minutes, and preferably for atleast about five minutes, during inoculation, improves efficiency formany applications. Alternative suitable techniques are well known.

[0043] After incubation, excess suspension is removed and stem segmentsare transferred to a co-cultivation medium. A suitable co-cultivationmedium comprises MS medium with about 0.4% glucose. According to apreferred embodiment, a phenolic compound, such as acetosyringone, isincluded in the co-cultivation medium at a concentration of at leastabout 1 μM and, preferably, at a concentration of about 5 μM. Otherphenolic compounds having similar structural and/or functionalproperties may additionally or alternatively be used. According to yetanother alternative embodiment, bud induction medium may be usedfollowing inoculation in the place of the co-cultivation mediumdescribed above. Suitable and preferred bud induction media preferablycomprise MS media with sucrose, BA, and NAA, and are described below.Co-cultivation preferably takes place with the explant stem segmentsplaced horizontally on the surface of the medium during a three-dayco-cultivation period.

[0044] Following the co-cultivation period, stem segments are removedfrom the medium and washed. A preferred washing medium comprises MSmedium with timentin, preferably at a concentration of 250 mg/l. Stemsegments are then cultured, preferably vertically, in a first selectionmedium. The first selection medium preferably comprises MS medium, acarbon source, a cytokinin and/or an auxin, timentin and a selectionagent. Suitable carbon sources include sucrose and /or glucose at aconcentration of from about 5 to 100 g/l. Sucrose at a concentration ofmore than about 2%, and preferably about 3%, is a preferred carbonsource. Suitable cytokinins include BA, Dimethylallylaminopurine (2iP),Kinetin (K) and Zeatin (Z) at a concentration of from about 0.1 to 10mg/l. Suitable auxins include NAA, Indoleacetic acid (IAA),Indolebutyric acid (IBA), and 2,4-dichlorophenoxyacetic acid (2,4-D) ata concentration of about 0.001 to 1 mg/l. The preferred concentration oftimentin is about 250 mg/l. The preferred selection agent is kanamycinat a concentration of about 50 mg/l. The choice and concentration of theselection agent will depend upon the selection marker introduced in thegenetic construct. The pH of the first selection medium is preferablyadjusted to about 5 to 6. Stem segments are subcultured in fresh mediumeach week for at least about 4 to 5 weeks until adventitious buds areproduced from the stem segments.

[0045] Putative transformed adventitious shoots are excised from thestem segments and transferred to shoot elongation medium or a secondselection medium. A preferred shoot elongation medium comprises fullstrength MS medium, sucrose at a concentration of about 30 g/l, BA at aconcentration of about 0.1 mg/l, NAA at a concentration of about 0.01mg/l, gibberellic acid at a concentration of about 1 mg/l, and aselection agent at a concentration greater than the concentration of theselection agent in the first selection medium. The choice andconcentration of the selection agent will depend upon the selectablemarker in the genetic construct. Kanamycin at a concentration of greaterthan 50 mg/l is a preferred selection agent for the second selectionmedium. Kanamycin at a concentration of about 100 mg/l is an especiallypreferred selection agent. Geneticin and neomycin are also suitableselection agents. The shoot elongation medium or second selection mediumalso preferably comprises Timentin at a concentration of about 250 mg/l.GUS staining of the stem segments of the shoots may also be monitored toeliminate chimeric shoots. This may be accomplished by taking crosssections of the basal regions of putative transformed shoots andstaining overnight according to methods described in Stomp,“Histochemical localization of β-glucuronidase,” in GUS Protocols: usingthe GUS gene as a reporter of gene expression, pp. 103-113, 1992. Toensure chimera-free transgenic plants, only the shoots showing 100% GUSstaining may be selected for plantlet development.

[0046] Transformed shoots are transferred to a suitable rooting medium.A preferred rooting medium comprises Gamborg medium (Sigma G5893) orKnop medium (Knop, Landw. Versuchs. Stat. 7:93-107, 1865) comprising IBAat a concentration of about 1 mg/l, a selection agent such as kanamycinat a concentration of about 100 mg/l, and timentin at a concentration ofabout 250 mg/l. Rooting is accomplished in a period of from about two tofour weeks and may involve an initial culture period in the dark toallow initial root development, followed by transfer to standardphotoperiod conditions. During elongation and rooting, explants may betransferred to larger culture vessels, such as Magenta boxes. Rootedshoots, or plantlets, may be transferred to a growth medium and grown tomature, genetically modified plants. Genetically modified plantsproduced according to the methods disclosed herein may be reproduced,for example, using standard clonal propagation techniques such asaxillary bud multiplication techniques.

[0047] The following examples are offered by way of illustration and notby way of limitation. Examples 1 to 3 describe experiments involvingoptimization of the regeneration and transformation protocols forEucalyptus transformation. All refer to adventitious bud induction fromstem segments obtained from in vitro micropropagated shoot cultures ofE. grandis x nitens clone 910.59 (Fletcher Challenge Forests, Ltd., NewZealand). The tissue culture conditions in all examples, unlessotherwise noted, were: temperature 20° C., 16 hour photoperiod, and coolwhite fluorescent lighting. These tissue culture conditions aregenerally preferred. All cultures were placed in 20×100 mm Petri dishes,10 stem segments per dish.

[0048] Plant Materials

[0049] All plant materials were provided by Te Teko laboratory, FletcherChallenge Forests Ltd., New Zealand. E. grandis X E. nitens clones910.59, 910.62 and 910.64 were obtained as in vitro micropropagatedshoot cultures grown on a multiplication medium (full strength MSmedium, sucrose 30 g/l, BA 0.1 mg/l, and NAA 0.01 mg/l) for 3 weeks, andthen transferred to elongation medium (full strength MS medium, sucrose30 g/l, BA 0.1 mg/l, NAA 0.01 mg/l and gibberellic acid 1 mg/l) for 4 to6 weeks.

[0050] Arobacterium Construct

[0051] The construction of the binary vector pKIWI is described byJanssen and Garner in Plant Molecular Biology 14:61-72, 1989. Thisconstruct contains a chimeric gene, which expresses β-D-glucuronidase(GUS) in transformed plant tissues but not in bacterial cells, since theGUS gene lacks a bacterial ribosome-binding site. Binary plasmid vectorpKIWI 105 was transformed into Agrobacterium tumefaciens strain AGL1(Biotechnology 9:963-967, 1991) by the freeze thaw method described inAn et al., “Binary vectors,” in Gelvin S B and Schilperoort R A, eds.,Plant Molecular Biology Manual, Dordrecht: Kluwer Academic Publishers,pp. A3/1-A3/19, 1988.

[0052] Preparation of Agrobacterium for Transformation

[0053] Colonies of Agrobacterium strain AGLI carrying pKIWI 105 weregrown on YEP medium (yeast 20 g, peptone 20 g, NaCl 10 g) for 2-3 days.A single colony from the plate was selected and grown in a culture tubecontaining 5 ml of YEP medium with kanamycin 100 mg/l and rifampicin 50mg/l. Cultures were incubated at 29° C. with vigorous shaking. Theovernight Agrobacterium culture was centrifuged at 3000 g for 20 minutesand resuspended with YEP; then washed 3 more times. The cells wereresuspended in {fraction (1/10)} sterile MS medium with the addition of50 μM acetosyringone and the OD₆₀₀ of the inoculum was adjusted toaround 0.15. Cultures were allowed to grow on a shaker for 3 to 4 hoursat 29° C. before inoculation.

[0054] Preparation of Plant Materials for Transformation

[0055] In vitro shoot cultures were subcultured in a multiplicationmedium (Full strength MS medium, sucrose at 30 g/l, BA at 0.1 mg/l, andNAA at 0.1 mg/l) for 3 weeks, and then transferred to shoot elongationmedium (fill strength MS medium, sucrose 30 g/l, BA 0.1 mg/l, NAA 0.01mg/l and gibberellic acid 1 mg/l) for 4 to 6 weeks. All cultures weretransferred to fresh medium 2-3 weeks before transformation.

[0056] Mature shoots of 3-4 cm were chosen. To maximize adventitious budinduction, only stem segments from the second and third nodes were used.All leaves were peeled and discarded. Additional wounding was achievedby light longitudinal cutting of both sides of the stem with a scalpelblade. Second and third nodes of the shoots were carefully excised andplaced in a flask containing the Agrobacterium suspension.

[0057] Inoculation of Explants with Agrobacterium and Regeneration ofPutative Transgenic Shoots

[0058] Stem segments were incubated with the Agrobacterium suspension,prepared as described above, for 30 minutes on a shaker at 100 rpm at20° C. After incubation, excess suspension was blotted with steriletissue papers, stem segments were then transferred to a co-cultivationmedium (MS medium with 0.4% glucose). All explants were placedhorizontally onto the surface of the medium during the 3 dayco-cultivation period. After co-cultivation, all stem segments wereremoved from the medium, washed with MS medium containing 250 g/Ltimentin 3 times for 5 minutes each. After washing, stem segments werecultured vertically in selection medium (MS+3% sucrose+cytokinin andauxin+timentin 250 mg/l+kanamycin 50 mg/l). In addition to kanamycin,selection of transformed tissues may be carried out using G-418, whichis also known as geneticin or neomycin. Other selection agentscorresponding to a selectable marker in the expression vector may alsobe used. Subculture of stem segments onto fresh medium was done everyweek for the first 4 weeks until adventitious buds were produced fromthe stem segments.

[0059] Selection of Stably Transformed Shoots

[0060] Putative transformed adventitious shoots were excised from thestem segments. All shoots were transferred to shoot elongation medium(MS medium, sucrose 30 g/l, BA 0.1 mg/l, NAA 0.01 mg/l, gibberellic acid1 mg/l, kanamycin 100 mg/l, timentin 250 mg/l) for 2 to 4 weeks. Thissecond step of selection medium with a higher kanamycin concentrationwas included to further eliminate chimeric shoots which might haveescaped from the first, lower kanamycin selection process. GUS stainingof the stem segments of the shoots was monitored to further eliminatechimeric shoots. This was done by taking cross sections of the basalregions of all putative transformed shoots and staining overnight withGUS (“Histochemical localization of β-glucuronidase,” in GUS protocols:using the GUS gene as a reporter of gene expression, pp. 103-113, 1992).Careful analysis of the GUS distribution patterns of the stem sectionsreveals shoots which are stably transformed (whole stem sections arestained blue) versus those which are partially blue, and those with nostaining at all. To ensure 100% chimera-free transgenic plants, onlythose shoots which showed 100% GUS staining were selected for plantletdevelopment.

EXAMPLE 1

[0061] This experiment was designed to determine the best age of stemsegments for adventitious bud induction. The basal medium used wasfull-strength Murashige and Skoog (MS) medium (Sigma M5519) and sucrose30 g/l. The age of stem segments (apical, 1st, 2nd, 3rd and 4th nodes)were tested in combination with the concentrations of Benzylaminopurine(BA-1, 2 and 3 mg/l). All media contained Naphthalene acetic acid (NAA)at 0.01 mg/l.

[0062] In vitro shoots of 3-4 cm were cut into single node segments. Theleaves were removed prior to culture. Approximately twenty explants werecultured for each treatment. Cultures were transferred to fresh mediaevery 3 weeks. Adventitious bud development assessments were done after5 weeks in culture. The percentages of explants forming adventitiousshoots for each treatment are listed below. BA Concentration Age of StemSegments 1 mg/l 2 mg/l 3 mg/l Apical Shoot Tips 25% (5/20)  5% (1/20)33% (7/20) First Node 57% (12/21) 35% (7/20) 20% (4/20) Second Node 76%(16/21) 12% (6/20) 20% (4/20) Third Node 75% (15/20) 15% (3/20) 15%(3/20) Fourth Node 45% (9/20) 38% (8/21) 10% (2/20)

[0063] To maximize the number of adventitious buds induced from stemsegments, only the 2nd and 3rd node segments were cultured on MS mediumcontaining 30 g/l sucrose, 1 mg/l BA and 0.01 mg/l NAA for 4 weeks insubsequent transformation experiments.

EXAMPLE 2

[0064] This experiment was designed to determine the optimalconcentration of kanamycin to be used for selection of transformed budtissues. Explants were cultured as described at the end of Example 1, incombination with kanamycin levels of 0, 5, 10, 25, 50 or 100 mg/l. Invitro shoots of 3-4 cm in size were cut into single node segments, andthe appropriate segments used. All leaves were removed prior to culture.Approximately thirty explants were used for each treatment. Cultureswere transferred to fresh medium after 3 weeks in culture, and thepercentage of bud inhibition determined after 5 weeks in culture.Kanamycin % of Stem Segments % Bud Inhibition Concentration FormingAdventitious Buds Relative To Control  0 mg/l 80% (24/30) 0%  5 mg/l 64%(18/28) 20%  10 mg/l 47% (14/30) 41%  25 mg/l 33% (10/30) 59%  50 mg/l15% (14/31) 81% 100 mg/l  3% (1/30) 96%

[0065] The highest degree of bud inhibition was found using 100 mg/lkanamycin. A lower level of selection (25-50 mg/l) will be used for theinitial selection of transformed adventitious buds. For the effectiveselection against chimeric shoots, 100 mg/l kanamycin will be used forthe subsequent selection of transformed shoots.

EXAMPLE 3

[0066] This experiment was designed to determine if a cytokininpretreatment of stem segments prior to Agrobacterium infection improvedthe tranformation efficiency. Second and third node segments wereprecultured on bud induction medium (MS medium containing 30 g/lsucrose, 1 mg/l BA and 0.01 mg/l NAA) for 0, 1, 2, 4 or 7 days prior toAgrobacterium infection.

[0067] In vitro shoots of 3-4 cm in size were cut into single nodesegments. A minimum of 50 nodal segments (2nd and 3rd nodes) were usedfor each treatment. Cultures were transferred to fresh media every 3weeks. All tissues were stained to detect GUS activity (Stomp,“Histochemical localization of β-glucuronidase,” in GUS Protocols: usingthe GUS gene as a reporter of gene expression, pp. 103-113, 1992) as anindication of transformation. Duration of % of Total Explants % of TotalExplants Cytokinin Pretreatment GUS Positive GUS Positive in Buds 0 Days9% (10/109) 4% (4/109) 1 Day 3% (2/73) 0% 3 Days 5% (3/60) 0% 4 Days 5%(3/60) 0% 7 Days 4% (2/53) 0%

[0068] The above results indicated that preculture of explants withcytokinin did not improve the transformation efficiency. In fact,explants turned brown following Agrobacterium infection. Except asotherwise indicated in subsequent transformation experiments, stemsegments were infected with Agrobacterium immediately after excision.

EXAMPLE 4

[0069] A preferred transformation and regeneration protocol, based onthe previous Examples and the disclosure made herein, is as follows.

[0070] Preparation of in vitro Micropropagated Stocks:

[0071] Regular subculture of shoots in elongation medium every 3-4weeks.

[0072] Duration =3-4 weeks

[0073] Preparation of Agrobacterium for Transformation:

[0074] Prepare fresh Agrobacterium cultures, centrifuge overnightculture and grow in {fraction (1/10)} strength sterile MS medium, withthe addition of 50 μM acetosyringone for 3-4 hours to an OD 600 of0.3-0.5.

[0075] Duration 2 days.

[0076] Preparation of Plant Materials for Tranformation:

[0077] Select elongated shoots of 3-4 cm in height, growing in a shootelongation medium (MS medium containing 30 g/l sucrose, 0.1 mg/l BA,0.01 mg/l NAA and 1 mg/l Gibberellic acid =GA3) Duration=continual

[0078] Inoculation of Explants with Agrobacterium:

[0079] Incubate stem segments with Agrobacterium suspension for 30minutes on a shaker (100 rpm) at 20° C. Transfer stem segments to aco-cultivation medium.

[0080] Duration=3 days.

[0081] Induction of Putative Transformed Adventitious Buds:

[0082] Wash explants 3×5 minutes with MS medium containing 250 mg/ltimentin on shaker.

[0083] After washing, culture stem segments in bud induction/selectionmedium (MS medium containing 30 g/l sucrose, 1 mg/l BA, 0.01 mg/l NAA,50 mg/l kanamycin and 250 mg/l timentin). Subculture to fresh mediumweekly.

[0084] Duration=3-4 weeks.

[0085] Regeneration of Putative Transgenic Shoots:

[0086] Excise all putative transformed adventitious shoots and transferto shoot elongation medium (MS medium containing 30 g/l sucrose, 0.1mg/l BA, 0.01 mg/l NAA, 1 mg/lGA3, 100 mg/l kanamycin and 250 mg/ltimentin).

[0087] Duration =4-6 weeks.

[0088] Production of Transgenic Plants:

[0089] Select all GUS positive shoots and transfer to rooting medium(Gamborg or Knop medium containing 1 mg/l Indole-3-butyric acid=IBA, 100mg/l kanamycin and 250 mg/l timentin)

[0090] Duration=2-4 weeks)

[0091] The total duration of transformation and regeneration procedureis about 10-15 weeks.

EXAMPLE 5 Transgene Expression in Transformed Tissues

[0092] The basic protocol for Eucalyptus explant production,longitudinal wounding and Agrobacterium infection as described inExample 4 was followed, except that the Agrobacterium strain AGL1contained pKIWI105 modified by the addition of a second Cauliflowermosaic virus 35S promoter fragment (positions −89 to 291) to yield adouble 35S promoter driving the reporter GUS gene. Followingco-cultivation, explants were cultured on bud induction/selection mediumfor a minimum of 4-5 weeks, after which explants were removed fromculture and stained to detect GUS activity (Stomp, “Histochemicallocalization of β-glucuronidase.” in GUS protocols: using the GUS geneas a reporter of gene expression, pp. 103-113, 1992). Blue-staining wasobserved in the shoot buds, showing that the shoot buds wereGUS-positive. This indicates the transformed nature of the shoots

EXAMPLE 6 Detection of Stable Integration of DNA by PCR

[0093] In addition to GUS staining, indication of stable integration offoreign DNA into plant DNA was carried out by using the Polymerase ChainReaction (PCR) with genomic DNA as a template. DNA was isolated from ablue-stained Eucalyptus shoot bud using CTAB (HexadecyltrimethylammoniumBromide) according to standard protocols.

[0094] Genomic DNA isolated from the blue shoot bud was used as templatefor PCR with primers 35S-GUS-1F (SEQ ID NO. 1) and 35S-GUS-2R (SEQ IDNO. 2). Cycling conditions were 94° C.—1 min (1 cycle); 94° C.—1 min,55° C.—1 min, 72° C.—1 min (35 cycles); and 72° C.—5 min (1 cycle).Plasmid DNA containing the introduced gene construct was used as apositive control. The DNA fragments were separated by agarose gelelectrophoresis.

[0095] The band obtained with the PCR product from DNA from the blueshoot bud was identical in size to the band obtained from the controlplasmid DNA, indicating that the Eucalyptus DNA was transformed with theintroduced DNA. PCR reactions using blue shoot DNA and AgrobacteriumvirG gene primers were negative, indicating that contaminating bacterialDNA was not present in the Eucalyptus DNA sample.

EXAMPLE 7 Modifications to Explant Inoculation/Infection Protocol forTransformation

[0096] Eucalyptus nodes were produced as in Example 4, then wounded bypuncturing at least the outer cellular layer using a sterile 30½ gaugeneedle in the regions adjacent to and surrounding the axillary region ofeach node. Following wounding, explants were infected with Agrobacteriumcontaining the double 35S-GUS construct, as described in Example 5,followed by co-cultivation on bud induction medium without selection.After co-cultivation, explants were transferred to budinduction/selection medium for continued growth and development.Explants were stained for GUS activity as in Example 5 at 4-5 weekspost-infection. Treatment % of Total GUS-Positive Explants in BudsNon-transformed control 0% Needle wounded 9%

[0097] The above results indicate that needle wounding, as well asco-cultivation on bud induction medium, Yielded transformed shoot buds.

[0098] Eucalyptus nodes were placed onto bud induction medium withoutselection for 7 days, to allow some axillary shoot growth. Followingthis, the basal region of each developing axillary shoot (below thelower-most leaves) was wounded using a scalpel or needle and explantsplaced into a suspension of Agrobacterium, as described in Example 5. Inaddition, following placement in bacterial solution, the explants wereplaced under vacuum using a vacuum pump, for 7 minutes, followed byco-cultivation on MS+0.4% glucose medium and subsequent growth on budinduction/selection medium as described in Example 5. Explants werestained for GUS activity as in Example 5 at 4-5 weeks post-infection.Treatment % of Total GUS-Positive Explants in Buds Non-transformedcontrol 0% Scalpel wound + vacuum 5%

[0099] The above results indicate that wounding of axillary shoots frompre-induced Eucalyptus nodes can be used as a method of targetingexplant tissues for Agrobacterium infection and transformation.

EXAMPLE 8 Effect on Transformation of Acetosyringone in theCo-cultivation Medium

[0100] The inclusion of a phenolic compound, acetosyringone, in theco-cultivation medium was tested. Eucalyptus nodal sections wereinfected with Agrobacterium containing the double 35S-GUS construct, asdescribed in Example 5, and then allowed to co-cultivate on MS+0.4%glucose medium containing acetosyringone, as described in Example 5.Following co-cultivation, explants were placed onto budinduction/selection medium for subsequent growth. Explants were stainedfor GUS activity as in Example 5 at 4-5 weeks post-infection.Co-Cultivation Treatment % of Total GUS-Positive (Acetosyringone)Explants in Buds 0 μM 5% 5 μM 8%

[0101] The above results indicate that the inclusion of 5 μMacetosyringone in the co-cultivation medium enhances transformationefficiency.

[0102] Bud induction medium was tested as an alternative toco-cultivation medium comprising MS+0.4% glucose. Eucalyptus nodalsections as described in Example 5 were used for Agrobacteriuminfection. During the infection, explants were infected under vacuum for7 minutes. Explants were then placed on MS+0.4% glucose or bud inductionmedium without a selection agent for the co-cultivation period, followedby culture on bud induction/selection medium, as described in Example 5.Explants were stained for GUS activity as in Example 5 at 4-5 weekspost-infection. Co-Cultivation Treatment % of Total GUS-PositiveExplants in Buds MS + 0.4% glucose 5% Bud induction medium 8%

[0103] The above results indicate that the use of bud induction mediumduring co-cultivation enhanced transformation efficiency.

EXAMPLE 9

[0104] An alternative preferred transformation and regenerationprotocol, based on the previous examples and the disclosure made herein,is as follows.

[0105] Preparation of in vitro Micropropagated Stocks:

[0106] Regular subculture of shoots in elongation medium every 3-4weeks.

[0107] Duration=3-4 weeks

[0108] Preparation of Agrobacterium for Transformation:

[0109] Prepare fresh Agrobacterium cultures, centrifuge overnightculture, and grow in {fraction (1/10)} strength sterile MS medium, withthe addition of 50 μM acetosyringone for 3-4 hours to an OD₆₀₀ of0.3-0.5.

[0110] Duration=2 days

[0111] Preparation of Plant Materials for Transformation:

[0112] Select elongated shoots of 3-4 cm in height, growing in a shootelongation medium (MS medium containing 30 g/l sucrose, 0.1 mg/l BA,0.01 mg/l NAA, and 1 mg/l Gibberellic acid=GA3). Cut the shoots intosingle node stem segments. Second and third note segments are preferredfor inoculation. Optionally, place the single node stem segments on budinduction medium for at least 3-4 days to stimulate some axillary shootgrowth.

[0113] Duration=continual

[0114] Inoculation of Explants with Agrobacterium:

[0115] Remove any leaves and wound the single node stem segments bypuncturing at least the outer cellular layer in a region adjacent to orsurrounding the axillary region of each node. Incubate stem segmentswith Agrobacterium suspension for 30 minutes on a shaker (100 rpm) at20° C. Apply a vacuum during at least a portion of the inoculationperiod. Transfer stem segments to a co-cultivation medium optionallycomprising a phenolic compound, such as acetosyringone.

[0116] Duration=3-10 days

[0117] Induction of Putative Transformed Adventitious Buds:

[0118] Wash explants 3×5 minutes with MS medium containing 250 mg/ltimentin on shaker. After washing, culture stem segments in budinduction/selection medium (MS medium containing 30 g/l sucrose, 1 mg/lBA, 0.01 mg/l NAA, 50 mg/l kanamycin, and 250 mg/l timentin). Subcultureto fresh medium weekly.

[0119] Duration=minimum 4-5 weeks

[0120] Regeneration of Putative Transgenic Shoots:

[0121] Excise all putative transfored adventitous shoots and transfer toshoot elongation medium (MS medium containing 30 g/l sucrose, 0.1 mg/lGA, 0.01 mg/l NAA, 1 mg/lGA3, 100 mg/kanamycin, and 250 mg/l timentin).

[0122] Duration=4-6 weeks

[0123] Production of Transgenic Plants:

[0124] Select all GUS positive shoots and transfer to rooting medium(Gamborg or Knop medium containing 1 mg/l Indole-3-butyric acid =IBA,100mg/l kanamycin, and 250 mg/l timentin).

[0125] Duration=2-4 weeks

[0126] SEQ ID NOS: 1-2 are set out in the attached Sequence Listing. Thecodes for nucleotide sequences used in the attached Sequence Listing,including the symbol “n,” conform to WIPO Standard ST.25 (1998),Appendix 2, Table 1. All references cited herein, including patentreferences and non-patent publications, are hereby incorporated byreference in their entireties. While in the foregoing specification,this invention has been described in relation to certain preferredembodiments, and many details have been set forth for purposes ofillustration, it will be apparent to those skilled in the art that theinvention is susceptible to additional embodiments and that certain ofthe details described herein may be varied considerably withoutdeparting from the basic principles of the invention.

1 2 1 23 DNA Artificial Sequence Made in a lab; derived from 35SCauliflower mosaic virus strain B29 promoter region 1 caaagatgtacccccaccca cga 23 2 25 DNA Artificial Sequence Made in a lab; derivedfrom 35S Cauliflower mosaic virus strain B29 promoter region 2ggctttcttg taacgcgctt tccca 25

We claim:
 1. A method for producing genetically modified plant materialof the Eucalyptus or Pinus species, comprising: culturing nodal stemsegments of a target plant selected from the Eucalyptus and Pinusspecies; transforming the stem segments with a genetic construct byincubating the nodal stem segments with an Agrobacterium culturetransformed with the genetic construct; promoting regeneration ofadventitious shoot buds from the transformed stem segments; selectingtransformed adventitious shoot buds; and regenerating transformed plantmaterial from the transformed adventitious shoot buds.
 2. A methodaccording to claim 1, wherein the stem segments are derived frommicropropagated shoot cultures.
 3. A method according to claim 1,wherein the target plant stem segments are pretreated in amultiplication medium and then transferred to a shoot elongation medium.4. A method according to claim 1, wherein the stem segments are woundedprior to incubation with the Agrobacterium culture.
 5. A methodaccording to claim 1, additionally comprising excising adventitiousshoots from the stem segments prior to selection of the transformedadventitious shoot buds.
 6. A method according to claim 1, wherein thegenetic construct comprises a selection marker that confers resistanceto a selection agent, the method additionally comprising selectingtransformed adventitious shoot buds by exposing the adventitious shootbuds to a first selection medium having a first concentration of aselection agent and subsequently exposing the adventitious shoot budssurviving exposure to the first selection medium to a second selectionmedium having a second concentration of a selection agent greater thanthe first concentration.
 7. A method according to claim 6, wherein thefirst and second selection media comprise kanamycin.
 8. A methodaccording to claim 7, wherein the first selection medium has aconcentration of kanamycin less than or equal to 50 mg/l, and the secondselection medium has a concentration of kanamycin greater than 50 mg/l.9. A method according to claim 1, wherein the genetic constructcomprises genetic material that is homologous to the genome of thetarget plant.
 10. A method according to claim 1, wherein the geneticconstruct comprises genetic material that is heterologous to the genomeof the target plant.
 11. A method according to claim 1, wherein thegenetic construct comprises genetic material that affects one of thefollowing phenotypic properties of the target plant: insect tolerance;disease resistance; herbicide tolerance; sterility; rooting ability;temperature tolerance; drought tolerance; salinity tolerance; woodproperties; and growth rates.
 12. A method according to claim 1, whereinthe genetic construct comprises genetic material encoding a polypeptideof interest or a functional portion of a polypeptide of interest.
 13. Amethod according to claim 1, wherein the genetic construct comprises anantisense copy of a gene or a portion of a gene encoding a polypeptideof interest or a functional portion of a polypeptide of interest.
 14. Amethod according to claim 1, additionally comprising exposing the stemsegments to a reduced pressure atmosphere during incubation with theAgrobacterium culture.
 15. A method according to claim 1, wherein thegenetic construct comprises at least one promoter region.
 16. A methodaccording to claim 1, wherein the genetic construct comprises more thanone promoter region.
 17. A method according to claim 1, additionallycomprising promoting axillary shoot growth prior to transforming.
 18. Amethod according to claim 1, comprising regenerating transformed plantmaterial by transferring the transformed adventitious shoot buds to arooting medium and forming plantlets.
 19. A method according to claim18, additionally comprising transferring the plantlets to a plantingmedium and growing the plantlets to form mature, genetically modifiedplants.
 20. Genetically modified plants produced according to the methodof claim
 19. 21. Plant materials and plants derived from the geneticallymodified plants of claim
 20. 22. Plant products derived from thegenetically modified plants of claim
 20. 23. Genetically modified plantmaterials produced according to the method of claim 1.